Characterization of Key Aroma Compounds in Raw and Thermally

Aug 3, 2016 - was to characterize the key odorants in freshly prepared blanched prawn meat (BMP) (Litopenaeus vannamei) by application of AEDA followe...
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Characterization of Key Aroma Compounds in Raw and Thermally Processed Prawns and Thermally Processed Lobsters by Application of Aroma Extract Dilution Analysis Veronika Mall and Peter Schieberle* Deutsche Forschungsanstalt für Lebensmittelchemie Lise-Meitner-Strasse 34, 85354 Freising, Germany ABSTRACT: Application of aroma extract dilution analysis (AEDA) to an aroma distillate of blanched prawn meat (Litopenaeus vannamei) (BPM) revealed 40 odorants in the flavor dilution (FD) factor range from 4 to 1024. The highest FD factors were assigned to 2-acetyl-1-pyrroline, 3-(methylthio)propanal, (Z)-1,5-octadien-3-one, trans-4,5-epoxy-(E)-2-decenal, (E)-3-heptenoic acid, and 2-aminoacetophenone. To understand the influence of different processing conditions on odorant formation, fried prawn meat was investigated by means of AEDA in the same way, revealing 31 odorants with FD factors between 4 and 2048. Also, the highest FD factors were determined for 2-acetyl-1-pyrroline, 3-(methylthio)propanal, and (Z)-1,5-octadien-3-one, followed by 4-hydroxy-2,5-dimethyl-3(2H)-furanone, (E)-3-heptenoic acid, and 2-aminoacetophenone. As a source of the typical marine, sea breeze-like odor attribute of the seafood, 2,4,6-tribromoanisole was identified in raw prawn meat as one of the contributors. Additionally, the aroma of blanched prawn meat was compared to that of blanched Norway and American lobster meat, respectively (Nephrops norvegicus and Homarus americanus). Identification experiments revealed the same set of odorants, however, with differing FD factors. In particular, 3-hydroxy-4,5-dimethyl-2(5H)-furanone was found as the key aroma compound in blanched Norway lobster, whereas American lobster contained 3-methylindole with a high FD factor. KEYWORDS: aroma extract dilution analysis, blanched prawn meat, fried prawn meat, Norway lobster, American lobster

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INTRODUCTION Over the past decades, crustaceans such as prawns, shrimp, lobsters, crayfish, or crabs have become more and more popular as healthy and tasty food. In particular, the characteristic fishy, roasty aroma of processed crustacean meat is highly appreciated by consumers all around the world. In former studies on cooked crustaceans, more than 450 volatiles have been identified. However, in earlier investigations, often a differentiation of odor-active compounds from the bulk of odorless volatiles, for example, by means of gas chromatography− olfactometry (HRGC-O), was not carried out in the respective food extract.1−6 Therefore, only some studies on the key odorants of processed crustaceans such as lobster, crab, and shrimp are available in which molecular data were correlated with sensory perception.7−13 Baek and Cadwallader12 analyzed the occurrence of key odorants in cooked meat of different crustacean species and reported 2-acetyl-1-pyrroline and 3(methylthio)propanal to be the main contributors to the overall aroma of cooked crustacean meat. In addition, they detected trimethylamine, 2,3-butanedione, 3-methyl-1-butanol, (Z)-4heptenal, 1-octen-3-one, 2-methyl-3-furanthiol, 2-acetylthiazole, and 2-acetyl-2-thiazoline as important odorants in cooked prawn and shrimp meat. In former studies on the aroma compounds of heated crustacean meat, also marine, leather-like, and dry seaweed-like odorants were detected, but their identification remained uncertain.7−9,11,12 Whereas Whitefield et al.14,15 found 2,6dibromophenol to be the origin of the iodine-like off-flavor of Australian prawns, Boyle et al.16,17 reported an abundant occurrence of bromophenols in fish and crustaceans and, contrary to former studies, they suggested that in low concentrations of approximately 0.05 ppb these halogenated © 2016 American Chemical Society

phenols might be responsible for the desirable brine-like, sealike flavor of fresh seafood. For the overall aroma of lobster meat, a slightly different set of key odorants was found. For example, different compounds, such as 2-acetyl-3-methylpyrazine, 2-acetylpyridine, 3-methylindole, (E)-2-octanal, and (E,E)-2,4-decadienal were reported.8,9 In a study using aroma extract dilution analysis (AEDA), the main odorants in roasted shrimp (Sergia lucens Hansen) were reported to be methanethiol, 1-pyrroline, N-(2′-methylbutyl)pyrrolidine, N-(3′-methybutyl)-pyrrolidine, methyl isopropyl disulfide, and 3-methylpyridine.13 Whereas the latter work was carried out on sun-dried seafood, the aim of this investigation was to characterize the key odorants in freshly prepared blanched prawn meat (BMP) (Litopenaeus vannamei) by application of AEDA followed by identification experiments using HRGC/MS and two-dimensional HRGC-GC/MS. To understand the influence of different processing methods on the formation of aroma compounds, the odorants of raw and fried prawn meat were compared to those in BMP. Finally, the influence of species of crustaceans was investigated by comparing the main odorants in blanched meat of two lobster types (Nephrops norvegius and Homarus americanus) to that of BMP. Received: Revised: Accepted: Published: 6433

June 17, 2016 August 3, 2016 August 3, 2016 August 3, 2016 DOI: 10.1021/acs.jafc.6b02728 J. Agric. Food Chem. 2016, 64, 6433−6442

Article

Journal of Agricultural and Food Chemistry

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Extraction of Raw Prawn Meat with Water and Dichloromethane. Minced raw prawn meat (250 g) was mixed with 400 mL of deionized water and stirred for 2 h at room temperature. This mixture was directly subjected to SAFE distillation24 and then extracted with dichloromethane (3 × 150 mL). After drying over anhydrous sodium sulfate, the organic layer was concentrated using the Vigreux column described above and subjected to HRGC-O and HRGC-GC-O/MS. Simultaneous Distillation/Extraction (SDE). To obtain better yields, a SDE according to the method of Nickerson and Likens27 was carried out additionally. For this extraction 200 g of powdered raw prawn meat was mixed with distilled water (800 mL) and heated. On the other side of the apparatus, dichloromethane was boiled at 45 °C. The SDE procedure was maintained for 2 h, and the aroma extract obtained was subsequently concentrated to ∼1 mL using Vigreux column distillation and further to ∼200 μL via microdistillation. HRGC-O, HRGC/MS, and HRGC-GC-O/MS. HRGC-O was performed by means of a gas chromatograph 5160 Mega series (Carlo Erba, Hofheim, Germany) with helium as carrier gas at a flow rate of 2.2 mL/min. The samples were applied by the cold-on-column technique onto the following fused silica capillaries: DB-FFAP (30 m × 0.25 mm i.d.; 0.25 μm film thickness) (J&W Scientific, Folsom, CA, USA) and DB-5 (30 m × 0.32; 0.25 μm film thickness) (MachereyNagel, Düren, Germany). After injection of the sample at 40 °C, the temperature was held for 2 min isothermally and then increased at 6 °C/min to 230 °C (DB-FFAP) or 240 °C (DB-5), respectively, and held for 5 min. The effluent of the capillary column was split 1:1 by volume and transferred to a flame ionization detector (FID) and a heated (200 °C) sniffing port made of alumina, respectively, using a Yshaped quick-seal glass connector (Chrompack, Frankfurt, Germany) and two deactivated fused silica capillaries (30 cm × 0.20 mm i.d. each). Linear retention indices (RI) were calculated from the retention times of n-alkanes as described previously.25 MS was performed by means of a Mat 95 S mass spectrometer (Finnigan, Bremen, Germany) connected to a 5160 Mega series gas chromatograph using the same capillaries as described above. Mass spectra in the electron impact mode (MS-EI) were generated at 70 eV. For two-dimensional HRGC−HRGC/MS applications, a Mega 2 series gas chromatograph (Fisons Instruments, Mainz-Kastel, Germany) was coupled to a CP 3800 gas chromatograph (Varian, Darmstadt, Germany) and a Saturn 2000 ion trap mass spectrometer (Varian) using the DB-FFAP capillary in the first and the DB-5 column in the second oven. The elution range selected was transferred into a cold trap (−100 °C) by means of a moving column stream switching system (MCSS) (Thermo, Dreieich, Germany) located in the first and a heated transfer line (250 °C) (Horst GmbH, Frankfurt, Germany) connecting both GCs. By heating the trap, the sample was transferred to the second column. Simultaneous sniffing and FID detection at the first HRGC or sniffing and MS detection at the second HRGC, respectively, were done by splitting the effluent of the respective capillary column as described above. Mass spectra in MS-EI mode were generated at 70 eV. AEDA. Three panelists performed HRGC-O with the undiluted aroma extract to confirm the perception of the whole set of aroma compounds within the sample and to minimize problems possibly caused by anosmia. Then, the AEDA was performed by one person to determine the flavor dilution (FD) factors of odor-active compounds.28 The original aroma distillate (200 μL) obtained from 50 g of crustacean meat was diluted stepwise using diethyl ether to obtain dilutions of 1:1, 1:2, 1:4, 1:8, 1:16, ..., 1:2048 of the original extract. Each dilution was analyzed by HRGC-O (injection volume = 1 μL) until no odor was detectable during GC-O. The same GC conditions and columns as described above were used.

EXPERIMENTAL PROCEDURES

Materials. Whiteleg shrimp (L. vannamei) (origin: aquaculture in Thailand), Norway lobster (N. norvegicus) (captured in the northeastern Atlantic (FAO-area 27) offshore Scotland), and American lobster tails (H. americanus) (captured in the northwestern Atlantic (FAOarea 21) offshore Canada) were purchased at a local supermarket. All three products were frozen in the raw state with shell. Chemicals. The reference compounds were obtained from the following sources: 2, 4, and 36 were gifts from Symrise (Holzminden, Germany); 5 (Alfa Aesar, Karlsruhe, Germany); 8−13, 15−17, 20− 23, 25−31, 34, 35, 38, 40−50, and U5 (Sigma-Aldrich Chemie, Taufkirchen, Germany); 14, 18, 19, and 37 (Fluka, Neu-Ulm, Germany); 24 (Chemos GmbH, Regenstauf, Germany); and 51 (Merck, Darmstadt, Germany). 2,4-Dibromophenol, dimethyl sulfate, and 2,4,6-tribromoanisole were obtained from Sigma-Aldrich Chemie. Reference solutions of (E,Z,Z)- and (Z,Z,Z)-5,8,11-tetradecatrien-2one were gifts from T. Hasegawa Co. (Tokyo, Japan). The following compounds were synthesized according to the literature cited: (Z)-3-methyl-1-butene-1-thiol (1) and (E)-2-methyl1-butene-1-thiol (3);18 2-acetyl-1-pyrroline (6);19 (Z)-1,5-octadien-3one (7),20 and 2,4,6-nonatrienal (32, 33);21 and (E)-3-heptenoic acid (39).22 Diethyl ether and dichloromethane were freshly distilled prior to use. Synthesis of 2,4-Dibromoanisole. Methylation of 2,4-dibromophenol yielded the respective dibromoanisole as described by McKillop et al.23 2,4-Dibromophenol (1 mmol, 0.25 g) was added to an aqueous solution of sodium hydroxide (10%, 0.5 mL) with stirring. After the addition of dimethyl sulfate (1 mmol, 0.126 g), the solution was first stirred at room temperature for 10 min and then refluxed for another 10 min to remove the remaining methylation reagent. After cooling, the aqueous solution was extracted with diethyl ether (total volume = 100 mL). The organic layer was washed with an aqueous solution of sodium hydroxide (5%, total volume = 50 mL) and water (total volume = 50 mL) and dried over anhydrous sodium sulfate. Characterization of 2,4-dibromoanisole was carried out by GC/ MS. MS-EI m/z (%): 63 (12), 75 (8), 170 (8), 172 (7), 221 (13), 223 (26), 225 (13), 249 (19), 251 (39), 253 (18), 264 (46), 266 (100), 268 (44). Cooking Process of Crustaceans (Blanching). After thawing, the crustaceans were cooked in unsalted boiling water (100 °C) for 1 min (whiteleg shrimp), 2 min (Norway lobster), or 4 min (American lobster tails), respectively. The shell was removed, and the meat was frozen with liquid nitrogen and minced to a fine powder. Frying Process of Prawns. After defrosting, prawns were panfried at 160 °C without using fat. After 6 min of evenly frying both sides, the prawns were cooled and the shells removed. The meat was frozen with liquid nitrogen and minced to a fine powder. Isolation of Volatiles; Separation into Acid and Neutral/ Basic Compounds. Meat powder (100 g) was mixed with anhydrous sodium sulfate (50 g) and extracted with diethyl ether (300 mL) with stirring for 2 h. After filtration, the volatiles were isolated by solventassisted flavor evaporation (SAFE).24 The aroma distillate obtained was then dried over anhydrous sodium sulfate and concentrated to ∼50 mL using a Vigreux column (60 cm × 1 cm). For fractionation, the distillate was extracted with an aqueous solution of sodium bicarbonate (0.5 mol/L; total volume = 150 mL). The organic layer was washed twice with brine (total volume = 150 mL) and dried over anhydrous sodium sulfate, yielding the neutral/basic fraction (NBF). The aqueous layers were recombined and adjusted to a pH of 2.5 using hydrochloric acid (2 mol/L), and the acidic compounds were extracted with diethyl ether (total volume = 300 mL). The combined ethereal solutions were dried over anhydrous sodium sulfate to yield the acidic fraction (AF). Both fractions were finally concentrated to 200 μL each using the Vigreux column described above followed by microdistillation at 40 °C.25 Isolation of Thiols. Thiols were isolated by means of mercurated agarose gel as described recently.26

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RESULTS AND DISCUSSION

Key Odorants in BPM. Volatiles from freshly prepared blanched prawns were extracted with diethyl ether and carefully isolated using SAFE.24 A small drop of the distillate when sniffed from a strip of filter paper evoked the characteristic 6434

DOI: 10.1021/acs.jafc.6b02728 J. Agric. Food Chem. 2016, 64, 6433−6442

Article

Journal of Agricultural and Food Chemistry

Figure 1. (A) FID chromatogram obtained by GC-O of an aroma distillate of blanched prawn meat with odorant areas perceived (FD ≥ 128). (B) FD chromatogram obtained by application of AEDA to an aroma distillate of blanched prawn meat (FD ≥ 64).

Figure 2. Structures of the most odor-active volatiles identified in blanched prawn meat (numbering refers to Table 1; FD factors in parentheses).

aroma of BPM, thus confirming that the entire set of aroma compounds had been isolated. Application of GC-O and AEDA to the distillate of BPM meat revealed 40 odor-active regions in the FD factor range from 4 to 1024. The highest FD factor was

obtained for the roasted, popcorn-like-smelling compound 6, followed by the cooked potato-like odorant 12 with an FD of 512 (Figure 1). Somewhat lower FD factors were found for compounds 7 (metallic, geranium-like), 36 (metallic), 39 (sour, 6435

DOI: 10.1021/acs.jafc.6b02728 J. Agric. Food Chem. 2016, 64, 6433−6442

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Journal of Agricultural and Food Chemistry

total of 20 odorants are reported for the first time in crustaceans among the 51 compounds listed in Table 1. In earlier studies, Kubota et al.35 and Kobayashi et al.36 had identified two new shrimp-like-smelling aroma compounds in cooked shrimp (Euphausia superba, Euphausia pacifica, and Sergia lucens Hansen) as stereoisomers of 5,8,11-tetradecatrien2-one. After synthesis of all eight possible isomers of the methyl ketone, the unknown odorants were characterized as (E,Z,Z)and (Z,Z,Z)-5,8,11-tetradecatrien-2-one.35,36 When the prawn meat was submitted to SDE, also in our study these isomers could be found by means of two-dimensional HRGC-GC-O/ MS and were identified by means of authentic reference compounds. However, no odor was perceivable at the sniffing port at the respective areas, suggesting that the odor thresholds of these methyl ketones were higher than their actual concentration in the BPM. Therefore, subsequent quantitative studies should evaluate their possible aroma contribution to the aroma of BPM. Influence of Different Thermal Processing Conditions on the Aroma Compounds in Prawn Meat. Prior to consumption, prawns are usually processed, and during heating a desirable roasted, sweet, and fishy aroma is developed. Although several odorants may be generated due to enzymatic reactions, the thermal formation of aroma compounds from odorless precursors plays an important role in the overall aroma of processed prawn meat.12,34 These precursors are located in the flesh as well as in the shell. To provide the full spectrum of aroma precursors, the prawns were processed as such. Whereas the aroma profile of the raw prawn meat was mainly dominated by metallic, cucumber-like, green, and fishy notes, it shifted to seasoning-like, fishy, and roasty odor notes after blanching (100 °C) (data not shown). During frying, the roasted, popcorn-like odor note strongly increased, whereas the fishy odor note somewhat decreased. To elucidate the reason for these sensory changes on a molecular basis, an AEDA was next carried out on the volatiles of fried prawn meat (FPM), using the same amount of prawn meat and following exactly the same workup procedure as for BPM. In FPM, 40 odorants were detected with FD factors between 4 and 2048, 38 of which were identified. Again, the highest FD factor was determined for 2-acetyl-1-pyrroline (6, roasted, popcorn-like), followed by 3-(methylthio)propanal (12, cooked potato-like) with an FD factor of 1024 and (Z)-1,5-octadien-3one (7, metallic, geranium-like) with an FD factor of 512 (Table 2). An FD factor of 256 was found for 4-hydroxy-2,5dimethyl-3(2H)-furanone (37, caramel-like, 4-HDF), (E)-3heptenoic acid (39, sour, fecal, moldy), and 2-aminoacetophenone (43, foxy, medicine-like), respectively. A somewhat lower FD factor of 128 was assigned to 2,3-diethyl-5methylpyrazine (13, earthy) and linalool (14, citrus-like, flowery). Among the compounds detected only in FPM, but not in BPM, were two roasty-smelling compounds 2furanmethanethiol (10), eliciting a coffee-like, roasted smell, and 2-acetylthiazole (21), with an earthy, roasted aroma note. Apart from some odorants with relatively low FD factors, basically the same set of odorants was identified in BPM and FPM, respectively, however with different FD factors for some compounds. Higher FD factors in FPM were determined for 4HDF, linalool, and 2,6-dimethoxyphenol, whereas the earthysmelling 2,3-diethyl-5-methylpyrazine (13, FD 128), γoctalactone (34, coconut-like, FD 64), γ-nonalactone (38, coconut-like, FD 32), and 2,6-dimethoxyphenol (46, smoky, FD 64) were not detected in BPM (Table 3). 2-Acetyl-1-

fecal, moldy), 43 (foxy, medicine-like), 11 (vinegar-like), 24 (fishy, green, straw-like), and 33 (fatty, oat-like) (Figure 1). Identification of the odorants was performed following the protocol described earlier.29 Comparison of retention indices on at least two columns with different stationary phases (DBFFAP and DB-5) and of the odor quality with the data of an inhouse database containing ∼800 food aroma compounds gave a first hint on possible structures of aroma compounds. Recording mass spectra of the analytes and comparison to retention indices, odor quality, and odor intensity of authentic reference compounds completed the identification. Following this approach, compound 6 was identified as 2-acetyl-1pyrroline and compound 12 as 3-(methylthio)propanal (Figure 2). Both compounds have also previously been described as aroma compounds in heated crustaceans.7−12,30 Odorants 7, 36, 39, and 43 with FD factors of 256 were identified as (Z)-1,5-octadien-3-one (metallic, geranium-like), trans-4,5-epoxy-(E)-2-decenal (metallic), (E)-3-heptenoic acid (sour, fecal, moldy), and 2-aminoacetophenone (foxy, medicine-like), respectively (Figure 2). (Z)-1,5-Octadien-3-one and 2-aminoacetophenone were also described by Lee et al.9 in lobster tail meat. Although (Z)-1,5-octadien-3-one was determined as a key odorant in freshly boiled trout,31 the foxy-smelling 2-aminoacetophenone was previously regarded to have a negative impact on the overall odor in fermented tuna sauce.32 trans-4,5-Epoxy-(E)-2-decenal and (E)-3-heptenoic acid were identified for the first time in crustaceans. Because (E)-3-heptenoic acid was not commercially available, it was synthesized closely following a reported procedure,22 and its mass spectrum is shown in Figure 3. To our knowledge this

Figure 3. Mass spectrum (MS-EI) of (E)-3-heptenoic acid isolated from blanched prawn meat.

compound was identified only in heated pork fat up to now and was proposed to be a precursor for γ- and δ-lactones.33 Moreover, the identification experiments revealed various compounds with FD factors of 128, for example, 1-octen-3one (5, mushroom-like), acetic acid (11, vinegar-like), 3methyl-2,4-nonanedione (24, fishy, green, straw-like), (E,E,Z)2,4,6-nonatrienal (33, fatty, oat-like), 3-hydroxy-4,5-dimethyl2(5H)-furanone (42, seasoning-like), decanoic acid (44, musty), 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone (45, seasoning-like), and indole (48, mothball-like) (Table 1). Whereas among the lipid-derived compounds, 1-octen-3-one34 was previously reported in heated lobster and shrimp,8−10,12 a 6436

DOI: 10.1021/acs.jafc.6b02728 J. Agric. Food Chem. 2016, 64, 6433−6442

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Journal of Agricultural and Food Chemistry Table 1. Most Odor-Active Volatiles (FD ≥ 4) in Blanched Prawn Meat RI ond a

no.

odorant

1 4 5 6 7 9 11 12 14 16 17 18 19 20 22 23 24 25 26 27 28 29 30 31 32 33 35 36 37 39 40 41 42 43 44 45 48 49 50 51

(Z)-3-methyl-1-butene-1-thiol (Z)-4-heptenal 1-octen-3-one 2-acetyl-1-pyrroline (Z)-1,5-octadien-3-one unknown acetic acid 3-(methylthio)propanal linalool 2-methylpropanoic acid (E,Z)-2,6-nonadienal 2-acetylpyridine butanoic acid phenylacetaldehyde 2- and 3-methylbutanoic acid (E,E)-2,4-nonadienal 3-methyl-2,4-nonanedione pentanoic acid (E,Z)-2,4-decadienal 2-acetyl-2-thiazoline (E,E)-2,4-decadienal geosmine unknown 2-methoxyphenol (E,Z,E)-2,4,6-nonatrienal (E,E,Z)-2,4,6-nonatrienal benzothiazole trans-4,5-epoxy-(E)-2-decenal 4-hydroxy-2,5-dimethyl-3(2H)-furanone (E)-3-heptenoic acid 4-methylphenol γ-decalactone 3-hydroxy-4,5-dimethyl-2(5H)-furanone 2-aminoacetophenone decanoic acid 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone indole 3-methylindole 2-phenylacetic acid 4-hydroxy-3-methoxybenzaldehyde

odor quality

b

fraction

roasted, sulfury fishy mushroom-like roasted, popcorn-like metallic, geranium-like citrus, green vinegar-like cooked potato-like citrus-like, flowery sweaty, fruity cucumber-like popcorn-like sweaty, cheese-like flowery, honey-like sweaty, fruity fatty, green fishy, green, straw-like sweaty fatty, green popcorn-like fatty beetroot-like metallic, green, fruity smoky, sweet fatty, oat-like fatty, oat-like leather-like, phenolic metallic caramel-like sour, fecal, moldy horse stable-like peach-like seasoning-like foxy, medicine-like musty seasoning-like mothball-like mothball-like, fecal honey-like, sweet vanilla-like

NBF NBF NBF NBF NBF NBF AF NBF NBF AF NBF NBF AF NBF AF NBF NBF AF NBF NBF NBF NBF NBF NBF NBF NBF NBF NBF AF AF NBF NBF AF NBF AF AF NBF NBF NBF NBF

c

FFAP

DB-5

FD factore

1024 1232 1288 1325 1371 1417 1442 1446 1532 1558 1578 1596 1614 1640 1660 1693 1715 1726 1748 1755 1800 1815 1837 1858 1863 1877 1949 2003 2029 2039 2068 2149 2200 2222 2260 2265 2458 2506 2569 2583

765 902 975 922 981 ndg 630 907 1098 792 1156 1031 825 1038 882 1215 1240 920 1291 1091 1314 1404 nd 1088 1262 1267 1219 1376 1067 1105 1073 1464 1104 1300 1376 1196 1290 1393 1268 1398

8 16 128 1024 256 4 128 512 4 8 32 64 32 8 64 32 128 4 4 64 64 16 8 64 4 128 64 256 32 256 4 32 128 256 128 128 128 16 32 32

earlier identified in Crustaceaef 1, 4, 6−12 8−10, 12 7−12, 30 9 30 7−9, 12 6 8−10 3

3 10, 12 9, 10

9

6, 9, 30 9 6

9

1−3, 6 9

a

The odorant was identified by comparing the retention indices on two stationary phases, mass spectra (MS-EI and MS-CI), and the odor quality with data of the respective reference compound. bOdor quality detected at the sniffing port at a dilution factor 5 times above the odor threshold of the reference compound. cFractions were obtained by separation of the neutral/basic and acidic volatiles. dRetention index determined in comparison to a homologous series of n-alkanes. eFlavor dilution factor: last dilution of an extract in which an odorant was still detectable. fAroma compound earlier identified in crustaceans according to the literature cited. gnd, not determined.

pyrroline, 3-(methylthio)propanal, and (Z)-1,5-octadien-3-one only showed any difference of one dilution step in their respective FD factors, which does not indicate a difference between blanched and fried prawn meat. However, quantitative data are necessary to provide evidence of their potential influence on the overall aroma of thermally processed prawn meat. On the other hand, for several compounds somewhat lower FD factors were determined in FPM as compared to BPM, for example, for the mushroom-like 1-octen-3-one (5), acetic acid (11, vinegar-like), 3-methyl-2,4-nonanedione (24, fishy, green, straw-like), 3-hydroxy-4,5-dimethyl-2(5H)-furanone (42, sea-

soning-like), and 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone (45, seasoning-like) (Table 3). Comparison with Raw Prawn Meat (RPM). To obtain information on thermal formation pathways of certain odorants, an additional AEDA was subsequently carried out on RPM, using the same amount of prawn meat and following the same workup procedure as for BPM and FPM. Whereas among the thermally generated aroma compounds, the amino acid degradation product 3-(methylthio)propanal showed the second highest FD factor in both thermally processed samples, it was not detectable in the raw prawn meat (Table 3). For 2acetyl-1-pyrroline, which is known to be generated by reaction of proline or ornithine with carbohydrates via the intermediate 6437

DOI: 10.1021/acs.jafc.6b02728 J. Agric. Food Chem. 2016, 64, 6433−6442

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Journal of Agricultural and Food Chemistry Table 2. Most Odor-Active Volatiles (FD ≥ 4) in Fried Prawn Meata RI ond

a

a

no.

odorant

2 3 4 5 6 7 8 10 11 12 13 14 15 17 18 19 20 21 22 23 24 25 26 27 28 31 33 34 35 37 38 39 42 43 44 46 47 49 50 51

3-methyl-2-butene-1-thiol (E)-2-methyl-1-butene-1-thiol (Z)-4-heptenal 1-octen-3-one 2-acetyl-1-pyrroline (Z)-1,5-octadien-3-one trimethylpyrazine 2-furanmethanethiol acetic acid 3-(methylthio)propanal 2,3-diethyl-5-methylpyrazine linalool unknown (E,Z)-2,6-nonadienal 2-acetylpyridine butanoic acid phenylacetaldehyde 2-acetylthiazole 2- and 3-methylbutanoic acid (E,E)-2,4-nonadienal 3-methyl-2,4-nonanedione pentanoic acid (E,Z)-2,4-decadienal 2-acetyl-2-thiazoline (E,E)-2,4-decadienal 2-methoxyphenol (E,E,Z)-2,4,6-nonatrienal γ-octalactone benzothiazole 4-hydroxy-2,5-dimethyl-3(2H)-furanone γ-nonalactone (E)-3-heptenoic acid 3-hydroxy-4,5-dimethyl-2(5H)-furanone 2-aminoacetophenone decanoic acid 2,6-dimethoxyphenol unknown 3-methylindole 2-phenylacetic acid 4-hydroxy-3-methoxybenzaldehyde

odor quality

b

pungent, sulfury roasted, sulfury fishy mushroom-like roasted, popcorn-like metallic, geranium-like earthy coffee-like, roasted vinegar-like cooked potato-like earthy citrus-like, flowery earthy, roasted cucumber-like popcorn-like sweaty, cheese-like flowery, honey-like earthy, roasted sweaty, fruity fatty, green fishy, green, straw-like sweaty fatty, green popcorn-like fatty smoky, sweet fatty, oat-like coconut-like leather-like, phenolic caramel-like coconut-like sour, fecal, moldy seasoning-like foxy, medicine-like musty smoky metallic mothball-like, fecal honey-like, sweet vanilla-like

fraction NBF NBF NBF NBF NBF NBF NBF AF AF NBF NBF NBF NBF NBF NBF AF NBF NBF AF NBF NBF AF NBF NBF NBF NBF NBF NBF NBF AF NBF AF AF NBF AF NBF NBF NBF NBF NBF

c

FFAP

DB-5

FD factore

1094 1105 1232 1288 1325 1371 1394 1428 1442 1446 1477 1532 1555 1578 1596 1614 1640 1643 1660 1693 1715 1726 1748 1755 1800 1858 1877 1918 1949 2029 2030 2039 2200 2222 2260 2275 2348 2506 2569 2583

821 819 902 975 922 981 1000 903 630 907 1156 1098 ndf 1156 1031 825 1038 1012 882 1215 1240 920 1291 1091 1314 1088 1267 1238 1219 1067 1359 1105 1104 1300 1376 1351 nd 1393 1268 1398

32 32 16 16 2048 512 8 8 32 1024 128 128 16 16 8 16 4 4 64 64 16 4 64 4 64 128 32 64 32 256 32 256 32 256 32 64 64 16 32 16

For footnotes a−e, see Table 1. fnd, not determined.

1-pyrroline and 2-oxopropanal,37 interestingly, the highest FD factor of 4096 was found in RPM (Table 3). Assuming that solvent extraction with subsequent SAFE distillation represents a very gentle method with only a minimum of thermal impact, artifact formation during workup can be excluded as the origin of this compound.24 However, Romanczyk et al.38 had shown for cocoa that 2-acetyl-1-pyrroline can also be formed microbiologically at low temperatures (35 °C) in the presence of the required precursors. Thus, a microbiological formation of this compound in RPM seems also likely. Other odorants known to be generated by thermally induced carbohydrate or protein degradation, such as 5-ethyl-3-hydroxy4-methyl-2(5H)-furanone, 4-HDF, or 3-hydroxy-4,5-dimethyl2(5H)-furanone, were found with higher FD factors in blanched or fried prawn meat as compared to raw prawn meat (Table 3). The caramel-like smelling 4-HDF had already been reported before as an odorant in cooked lobster tail meat.9

Typical lipid degradation products such as (Z)-4-heptenal, (E,E)-2,4-nonadienal, (E,Z)-2,6-nonadienal, and 1-octen-3-one were also determined with somewhat higher FD factors in prawn meat after heat treatment (Table 3). The roasty-smelling compound 2-acetylthiazole, which had been described as an important odorant in blanched crustaceans before,12 was not detected in BPM, and only a low FD factor of 4 was determined in FPM. Reasons might be the different crustacean species and different processing parameters used in the previous investigation. Origin of the Typical Marine, Sea Breeze-like Odor of RPM. Despite the distinct fresh, marine, and sea breeze-like odor particularly recognized in the aroma profile of raw prawn meat, AEDA and identification experiments did not reveal any compounds covering this odor attribute. To further investigate the origin of this typical odor, minced RPM was subjected to a subsequent water and dichloromethane extraction followed by 6438

DOI: 10.1021/acs.jafc.6b02728 J. Agric. Food Chem. 2016, 64, 6433−6442

Article

Journal of Agricultural and Food Chemistry

Table 3. Comparison of the FD Factors of Selected Odor-Active Volatiles in Raw Prawn Meat (RPM) as well as in Blanched (BPM) and Fried Prawn Meat (FPM) FD factor inc a

no.

odorant

6 12 7 13 14 34 46 38 45 4 23 37 17 5 24 42 11

2-acetyl-1-pyrroline 3-(methylthio)propanal (Z)-1,5-octadien-3-one 2,3-diethyl-5-methylpyrazine linalool γ-octalactone 2,6-dimethoxyphenol γ-nonalactone 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone (Z)-4-heptenal (E,E)-2,4-nonadienal 4-hydroxy-2,5-dimethyl-3(2H)-furanone (E,Z)-2,6-nonadienal 1-octen-3-one 3-methyl-2,4-nonandione 3-hydroxy-4,5-dimethyl-2(5H)-furanone acetic acid

odor quality

b

roasted, popcorn-like cooked potato-like metal, geranium-like earthy citrus-like, flowery coconut-like smoky coconut-like seasoning-like fishy fatty, green caramel-like cucumber-like mushroom-like fishy, green, straw-like seasoning-like vinegar-like

RPM

BPM

FPM

4096